Expression of epithelial-mesenchymal transition markers SNAI1 and ZEB1 results in transcriptional alterations that promote tumor progression and immune evasion in prostate cancer

Author(s)

Luiz Paulo Chaves de Souza

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-05-10

Examining board members

Jeremy Andrew Squire; Sandra Martha Gomes Dias; Juliana Meola Lovato

• Advisor: Jeremy Andrew Squire

Abstract

Prostate Cancer (PCa) is the second most common type of cancer among men. The course of the disease is usually favorable. However, some patients develop the most aggressive form of the disease, with a recurrence rate of 30% for patients treated with radical prostatectomy. These patients are unresponsive to androgen deprivation therapy and chemotherapy, which is a significant cause of morbidity and mortality. Despite the effectiveness of immunotherapy in other tumor types, the response rate in patients with the advanced form of PCa has been disappointing, approaching only 15%. Currently, the immune evasion mechanisms of PCa are not well understood, and there are no definitive biomarkers to stratify patients who will develop an aggressive form of the disease or to predict who will respond to immunotherapy. Recently it has been shown that PCa can reactivate an embryogenic program called Epithelial-Mesenchymal Transition (EMT), which is capable of contributing to increased invasion, migration, therapeutic resistance, and metastasis. The EMT process has previously been associated with immune evasion in other types of solid tumors, but in PCa, a definitive relationship between immune response and EMT has yet to be studied. In this project, we investigated the impact of SNAI1 and ZEB1 expression, known EMT markers, on the PCa transcriptional profile, especially on genes and pathways related to the immune response. A cohort of 51 PCa patients undergoing radical prostatectomy were used. Formalin-fixed, paraffin-embedded tumor samples were collected retrospectively. Regions of interest were marked, and adjacent sections were sent to the Ontario Institute of Cancer Research for RNA extraction and transcriptomic analysis using BC360, PanCancer Pathway, Immune profiling and Human V3 miRNA NanoString Panels. Differential expression and pathway enrichment analyses comparing patients with higher expression of the genes of interest were performed using the Immune Profiling panel. Validation was performed using the PanCancer panel Pathway and public domain TCGA data. The enriched miRNAs and their targets were investigated. TCGA data was also used to perform digital cytometry analysis using the CIBERSORT algorithm. SNAI1 was associated with elevated pathological staging and ZEB1 with a lower probability of recurrence-free survival. Both markers were associated with the expression of genes related to greater aggressiveness, such as ETS1, COL3A1 and RUNX1, and to immune evasion such as CTLA4 and PD-L1. Complementarily, pro-inflammatory and pro-tumorigenic pathways, such as inflammatory response and TNF signaling via NF- kB were enriched in the SNAI1 high group. Analysis using TCGA data also showed enrichment of complement, interferon-gamma and IL6/JAK/STAT3 signaling pathways. The ZEB1 high expression group was associated with a greater number of enriched miRNAs, among which six target the EMT pathway itself, which suggests a negative feedback mechanism elicited by the expression of this gene. TCGA data also showed that both genes are associated with greater expression of immunological checkpoints, and the analysis of digital cytometry showed that the expression of both markers is associated with an enrichment of quiescent cells, such as naïve B lymphocytes, in the tumor microenvironment. It was also found that high expression of ZEB1 was associated with a depletion of effector cells, such as CD8 lymphocytes, and a M0-M2 polarization of tumor-associated macrophages. Collectively, these data suggest that EMT is associated not only with greater tumor aggressiveness but also with mechanisms of immune evasion, which could partially explain the poor response of PCa to immunotherapy. It is suggested that the genes identified as being involved in immune pathways in this project may be promising expression biomarkers for the future use of checkpoint inhibitor drugs in PCa. (AU)

• FAPESP's process: 20/12816-9 - Does ZEB1 cooperate with PTEN loss and TMPRSS2-ERG fusion in prostate cancer immune evasion?
• Grantee: Luiz Paulo Chaves de Souza
• Support Opportunities: Scholarships in Brazil - Master